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冷适应蛋白(cold-regulated,COR413)基因作为植物特有的一类低温胁迫响应功能基因,在抗寒过程中发挥重要作用。为了鉴定葡萄属(Vitis)植物COR413基因参与低温胁迫响应的功能特点,本研究基于欧洲葡萄基因组数据库挖掘,采用同源克隆法获得了抗寒种质山葡萄(Vitis amurensis)COR413基因家族成员的4条序列,分别命名为Va COR413-PM2A、-PM1X1、-IM1与-PM2B。经序列分析表明,所克隆的4条山葡萄COR413基因家族成员与欧洲葡萄相应基因的核苷酸与氨基酸序列高度同源,其核苷酸序列的差异仅为个别单核苷酸的替代。所克隆的4个山葡萄冷适应蛋白家族基因均含有WCOR413保守结构域、跨膜结构域与丝/苏/酪氨酸磷酸化位点,其中仅Va COR413-PM1X1含有一个预测的N端信号肽序列,仅Va COR413-PM2B包含1个糖基化磷脂酰肌醇锚点。源于2个葡萄种质中COR413基因家族成员可以聚类为2个明显的簇,COR413-PM2A,-PM1X1与-PM2B聚类在一簇,而COR413-IM1分布在另外一簇。半定量RT-PCR组织特异性检测结果表明,Va COR413-PM2A、-PM1X1、-IM1与-PM2B在山葡萄不同组织和器官中表达存在差异,且不同于欧洲葡萄相应基因的表达模式;其中山葡萄Va COR413-PM2A、-PM1X1、-IM1与-PM2B在所有检测的组织中均表达且强度不同,Va COR413-PM2A与-PM1X1在嫩梢、卷须、花絮以及幼叶中表达量较高。在低温胁迫条件下,山葡萄COR413基因家族4个成员参与低温胁迫的表达模式分为2类,而欧洲葡萄中相应成员有3种模式;山葡萄Va COR413-IM1受低温胁迫24 h表达量最高,为对照的9.57倍,而欧洲葡萄Vv COR413-IM1在低温胁迫3 h后的表达量较高,约为对照的3.65倍。低温胁迫下的表达数据表明,山葡萄COR413基因家族成员中聚类为一簇的Va COR413-PM2A,-PM1X1与-PM2B响应低温总体呈下调表达,其表达幅度均低于欧洲葡萄相应基因的表达量,而聚类在另外一簇的Va COR413-IM1对低温胁迫(24 h)响应诱导表达显著;研究结果为探明该基因家族成员在葡萄响应低温胁迫过程中的可能功能和作用机制提供了参考依据,同时为葡萄属植物抗寒(冻)性分子改良提供了理论依据。
Cold-regulated (COR413) gene, as a plant-specific cold-stress response gene, plays an important role in cold-resistance. In order to identify the functional characteristics of COR413 gene in response to low temperature stress in Vitis plant, we cloned four genotypes of Vitis amurensis COR413 gene family using homologous cloning method based on the European grape genome database. The sequences were named Va COR413-PM2A, -PM1X1, -IM1 and-PM2B, respectively. Sequence analysis showed that the nucleotide and amino acid sequences of COR413 gene family members of four clones were highly homologous to the corresponding genes of European grapevines, and the difference of nucleotide sequences was only an alternative single nucleotide. All four cloned cold-adapted protein family genes contained WCOR413 conserved domain, transmembrane domain and filament / threo / tyrosine phosphorylation sites, of which only Va COR413-PM1X1 contained a predicted N-terminal signal peptide sequence , Only Va COR413-PM2B contains one glycosylated phosphatidyl inositol anchor. COR413 gene family members from two grape germplasms can be clustered into two distinct clusters. COR413-PM2A, -PM1X1 and -PM2B cluster in one cluster, while COR413-IM1 distribute in another cluster. The results of tissue-specific RT-PCR showed that the expression of Va COR413-PM2A, -PM1X1, -IM1 and -PM2B was different in different tissues and organs of grapevine, and different from the expression patterns of corresponding genes in grapevine in Europe. Among them, COR413-PM2A, -PM1X1, -IM1 and -PM2B were expressed in all the tested tissues with different intensities. Va COR413-PM2A and -PM1X1 expressed more in shoots, tendrils, taffeta and young leaves. Under low temperature stress, four members of COR413 gene family were involved in the two types of low temperature stress, while the corresponding members of European grape had three patterns. Va COR413-IM1 was the highest expression at 24 h 9.57 times of the control, while the expression level of Vv COR413-IM1 in European grapevine after 3 h of low temperature stress was about 3.65 times that of the control. Under low temperature stress, the expression data of Va COR413-PM2A, -PM1X1 and -PM2B in clusters of COR413 gene family were all down-regulated in low temperature and the expression amplitude was lower than that of the corresponding genes in European grape , Whereas Va COR413-IM1 clustered in another cluster induced significant response to low temperature stress (24 h). The results provide a reference for exploring the possible function and mechanism of this gene family in response to low temperature stress in grapevine Based on the same time for the grape plants cold (freezing) molecular improvement provides a theoretical basis.